High Fidelity (HF®) Restriction Enzymes have 100% activity in CutSmart™ Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity. HF enzymes are all Time-Saver™ qualified and can therefore cut substrate DNA in 5-15 with the flexibility to digest overnight without degradation to DNA. Engineered with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while offering flexibility in experimental design.
NEB extensively performs quality controls on all standard and high-fidelity (HF®) restriction enzymes. Examples of nuclease contamination studies for some of our HF restriction enzymes are shown below.
Product SourceAn E. coli strain that carries the cloned and modified BsaI gene from Bacillus stearothermophilus 6-55 (Z. Chen)
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|Gel Loading Dye, Purple (6X)||25||6X|
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme required to digest 1 µg of pXba DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
1X CutSmart® Buffer
Incubate at 37°C
1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C
Activity in NEBuffersNEBuffer 1.1: 50%
NEBuffer 2.1: 100%
NEBuffer 3.1: 25%
CutSmart® Buffer: 100%
10 mM Tris-HCl
200 mM NaCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
pH 7.4 @ 25°C
Heat Inactivation65°C for 20 min
dam methylation: Not Sensitive
dcm methylation: Blocked by Overlapping
CpG Methylation: Blocked by Some Combinations of Overlapping
Quality Control AssaysThe following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
- Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
- Exonuclease Activity (Radioactivity Release):
The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
- Ligation and Recutting (Terminal Integrity):
After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
- Non-Specific DNase Activity (16 hour):
The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at email@example.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Gel Loading Dye, Purple (6X)
- Is there a difference in cutting close to the ends between BsaI-HF and BsaI?
- How does the level of star activity of BsaI-HF compare to BsaI?
- What does HF® refer to following the name of a restriction enzyme?
- When is star activity a concern?
- Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
- When should I choose the High Fidelity (HF®) version of the enzyme?
- How is the improvement in fidelity of HF restriction endonucleases quantitated?
- What is the Fidelity Index (FI)?
- Can the change in buffer preference of the HF enzyme be advantageous?
- Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
- What does it mean to be Time-Saver™ qualified?
- What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
- Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
- How can I access the old NEBuffer™ Activity Chart?
- I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason?
- Which restriction enzymes are used in Golden Gate Assembly?
- Which restriction enzymes are used in GoldenBraid Assembly?
- Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as SYBR® and GelRed™ during gel electrophoresis?
- Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
- Why is my Restriction Enzyme not cutting DNA?
- Why do I see a DNA smear on an agarose gel after a restriction digest?
- Why do I see additional DNA bands on my gel after a restriction digest?
- How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
- Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?
- Alphabetized List of Recognition Specificities
- Buffer and Diluent Formulation Table
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Cross Index of Recognition Sequences
- Dam-Dcm and CpG Methylation
- Enzymes with Nonpalindromic Sequences
- Frequencies of Restriction Sites
- Type IIS Restriction Enzymes
- Why Choose Recombinant Enzymes?
Usage Guidelines & Tips
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Activity of Restriction Enzymes in PCR Buffers
- Cleavage Close to the End of DNA Fragments
- Cleavage of Supercoiled DNA
- Dam and Dcm Methylases of E. coli
- Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
- Double Digests
- Heat Inactivation
- Megabase Mapping
- NEBuffer™ Activity/Performance Chart with Restriction Enzymes
- Optimizing Restriction Endonuclease Reactions
- Reduced Star Activities of HF® Enzymes
- Restriction Endonucleases - Survival in a Reaction
- Restriction Enzyme Diluent Buffer Compatibility
- Restriction Enzyme Tips
- Single Letter Codes
- Star Activity
- Traditional Cloning Quick Guide
- Abil Z, Denard CA, Zhao H (2014). Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA J Biol Eng. 8(1), 7. PubMedID: 24581042, DOI: 10.1186/1754-1611-8-7
- Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T (2014). Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system Sci Rep. 4, 5400. PubMedID: 24954249, DOI: 10.1038/srep05400
- Binder A, Lambert J, Morbitzer R, Popp C, Ott T, Lahaye T, Parniske M (2014). A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants PLoS One. 9(2), e88218. PubMedID: 24551083, DOI: 10.1371/journal.pone.0088218
- Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology Plant Physiology. 162(3), 1618-31. PubMedID: 23669743